Combination comprising N-acetyl-L-cysteine and its use

ABSTRACT

A combination of N-acetyl-L-cysteine, selenium in the form of selenomethionine and melatonin, and a medical product or pharmaceutical composition comprising such combination, useful for the treatment of a variety of diseases and conditions is described. The combination of N-acetyl-L-cysteine, selenium in the form of selenomethionine and melatonin is also useful for cosmetic treatment of skin and as an antibacterial agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of Application No. PCT/EP2012/054360filed Mar. 13, 2012, which claims priority from Swedish Application No.1100238-3 filed on Apr. 1, 2011, U.S. Provisional Application No.61/471,162 filed on Apr. 3, 2011, Swedish Application No. 1150696-1filed on Jul. 15, 2011, and U.S. Provisional Application No. 61/508,262filed on Jul. 15, 2011, the contents of each of which is herebyincorporated herein in its entirety by express reference thereto.

TECHNICAL FIELD

The present invention relates to a combination of N-acetyl-L-cysteine,selenium, such as selenomethionine, and melatonin, and to apharmaceutical composition comprising such combination, useful for thetreatment of a variety of diseases and conditions, e.g., benign andmalignant neoplasia including various types of cancers, autoimmunediseases, neurodegenerative diseases, endocrinological diseases, type 2diabetes, all types of fibrosis, amyloidosis, endometriosis, polycysticovary syndrome, dysmenorrhea, and dermatological diseases includingvitiligo, alopecia and psoriasis. The combination is also useful as anantibacterial agent and for cosmetic purposes.

BACKGROUND

Control and modulation of intracellular reduction and oxidation (redox)environment is of fundamental relevance for cellular processes. Forexample, loss in the redox control of the cell cycle can lead to a moreoxidant environment, promoting the progression from G1 to S phase andtherefore leading to aberrant proliferation, a hallmark of variousneoplasies. Also, most chronic inflammatory diseases are accompanied bya loss of redox control. Dysregulation towards a more oxidizedintracellular environment is thus associated with aberrant proliferationand inflammation and is therefore ultimately related to diseases suchas, but not limited to, cancer, neurodegenerative disease, diabetes,aberrant wound healing, and fibrosis. In addition, in oxidativeenvironments, proteins can be oxidative modified, a process oftenfollowed by the formation of aggregates, also in the form of amyloids.

The functional status of cells is under the control of external stimuli,affecting the function of critical proteins and gene expression. Signalsensing and transduction by messengers to specific effectors operate bypost-translational modification of proteins, among which thiol (—SH)reduction/oxidation (redox) switches play a fundamental role. Sensitivecysteine residues in proteins constitute the mentioned redox switches,where “sensitive” indicates a lower electrochemical potential of thecysteine/cystine redox couple (SH/S—S) resulting from a specific proteinconformation. Thiol redox switches in for example enzymes, receptors,transporters, transcription factors, structural elements, regulators ofprotein trafficking synthesis and degradation, and the cytoskeletalstructure all deeply affect the overall cellular homeostasis.

The production of oxidants in mammalian cells derives from fundamentalprocesses such as glycolysis and mitochondrial respiratory chain, endingup with the production of reactive oxygen species, among them the moststable is represented by hydrogen peroxide (H₂O₂). The produced oxidantsmust be under a strict control and a reversal of the oxidative pathwayis necessary. The redox control is affected by several low molecularweight substances and a complex network of enzymes, including amongothers, glutathione reductase (GR) and the family of selenium-dependentglutathione peroxidases (GPx). Reduced glutathione (GSH) is a dominantlow molecular weight thiol species in most organisms, whose redoxpotential is used by the many enzymes in charge of the redox control incells, including GR and GPx, to recycle their own redox status.

Reduced glutathione (GSH) is a tripeptide present in all human tissuesat relatively high concentrations, even above 10 mM. It has manyimportant functions in the body. As described above, it can be regardedas the major endogenous antioxidant, participating: 1) in the control ofthe cell redox status; and 2) in the control of the oxidation status ofproteins relevant in signaling, including the receptors of externalstimuli (e.g. hormone receptors). It plays a fundamental role innumerous metabolic and biochemical reactions such as DNA synthesis andrepair, protein synthesis, prostaglandin synthesis, amino acidtransport, and enzyme activation. It also participates in the regulationof the nitric oxide cycle, detoxifies many carcinogens and otherxenobiotics and has an essential role for optimal response in many partsof the immune system. Thus, most systems in the body can be affected bythe state of the glutathione system. For instance, states of glutathionedeficiency include HIV/AIDS, chemical and infectious hepatitis, prostateand other cancers, cataracts, Alzheimer's disease, Parkinson's disease,chronic obstructive pulmonary disease, asthma, radiation poisoning,malnutritive states, arduous physical stress and aging, and has beenassociated with suboptimal immune response. Low glutathione is alsostrongly implicated in wasting and negative nitrogen balance, as seen incancer, AIDS, sepsis, trauma, burns and even athletic overtraining, aswell as in bipolar disorder, major depressive disorder, andschizophrenia.

Glutathione is synthesized from the amino acids L-cysteine, L-glutamicacid and glycine. The sulfhydryl (thiol) group (SH) of the cysteineserves as a proton donor and is responsible for the biological activityof glutathione. The supply of cysteine is the rate-limiting factor inglutathione synthesis by the cells, since cysteine is a relatively rarenutrient. Glutathione supplementation has thus been suggested forvarious diseases and symptoms.

However, glutathione taken orally can be degraded already in the stomachand is not well absorbed across the gastrointestinal tract. Thus raisingglutathione levels through direct supplementation of glutathione isdifficult. Instead supplements of agents that serve as glutathioneprecursors are used to increase the plasma concentration of glutathione.N-acetyl-L-cysteine (NAC in the following) is a simpler molecule thanglutathione, diffuses freely in almost all tissues and cells and is themost bioavailable precursor of glutathione. Among the several thiolagents tested for their efficacy in modulating cellular redox status,NAC holds most promise for human use. A relevant advantage in theclinical use of NAC is the virtual absence of side effects. Thiscompound has been long available for the clinic as a mucolytic agent andas an antidote after paracetamol poisoning.

In recent years NAC has also been acknowledged as having otherbeneficial properties. For example, NAC has been reported to have ananti-inflammatory effect and has been added to the family ofnon-steroidal anti-inflammatory drugs (NSAIDs). The inventors of thepresent disclosure have previously shown that in mammalian cells, invitro, NAC inhibits proliferation and promotes quiescence, furtherevolving in terminal differentiation (WO 02/051405 A1, T. Parasassi, et.al. (Cell Death and Differentiation (2005), Vol. 12, No. 10, pages1285-1296); E. K. Krasnowska et. al. (Free Radicals Biology and Medicine2008, 45(11):1566-72) and A. C. Gustafsson et. al. (BMC Cancer (2005),5:75). NAC has in this context been found to possess a markedantiproliferative effect on cancer cells and has also been found to beeffective in the treatment of endometriosis. Additionally it has beenused in the treatment of polycystic ovary syndrome (PCOS) as well as fortreatment of various other diseases and conditions, e.g. as anephroprotective agent, interstitial lung disease, schizophrenia,bipolar disorder and depression, and has been suggested for variousother uses.

Thus, NAC is effective for many uses. Nevertheless, prolonged NACtreatments result in a decreased plasma level of NAC, so that itseffective concentration must be increased and the risk of undesired sideeffects accordingly increases (L Pendyala and P J Creaven, CancerEpidemiol Biomarkers Prev 1995; 4:245-251). In order to counteract thedeclining effect of NAC, pulsed treatments were proposed, to allow for awashout period. Another possible solution would be of reducing theconcentration at which NAC is effective, which would be advantageousalso for short term treatments.

There has been a wide recent interest in the protecting and/ortherapeutic role of both selenium (Se in the following) and melatonin(Mel in the following). Se supplementation has for example beensuggested for prevention of cancer, as an antioxidant or immuneenhancer. Likewise, melatonin has been studied for the treatment ofcancer, immune diseases and various other disorders. Despite this,controversial effects of these two substances were also reported. Forinstance, Mel has been acknowledged for its antioxidant action, but wassometimes also reported to act as a pro-oxidant (Cemeli E, BaumgartnerA, Anderson D. Mutat Res. 2009; 681:51-67; Wölfler A, Caluba FTC, AbujaP M et al. FEBS Lett. 2001; 502(3):127-31; Radogna F et al. Toxicologyand Applied Pharmacology 2009; 239:37-45).

Regarding Se, a large trial showed that the expected prevention ofprostate cancer or of other cancers was not achieved (Lippman S M etal., Journal of American Medical Association. 2009; 301(1):39-51).EAKlein (J Natl Cancer Inst. 2009; 101: 283-285) concluded with thecautionary lesson that “well-performed large-scale controlled trials donot always validate what we believe biology indicates and that our modelsystems are imperfect measures of clinical outcomes in the real world.”Also, although a preventive role of Se on the risk of diabetes wasreported and ascribed to its “insulin-like” activity and to theantioxidant properties of the selenoenzymes, a prospective study did notreport any significant relationship between selenium and the risk ofdiabetes (Akbaraly T N et al., Nutr Metab (Lond). 2010; 7:21).

Balansky et al., “Interactions between N-acetylcysteine and sodiumselenite in modulating the clastogenicity of urethane and2-acetylaminofluorene in mice”, Int. J. Cancer, Vol. 108, 2004, pp.158-161, discloses the use of a combination of NAC and Se forattenuating the adverse effects of cytotoxic drugs and chemopreventiveagents in the treatment of cancer.

Safarinejad et al., “Efficacy of selenium and/or N-acetyl-cysteine forimproving semen parameters in infertile men: a double-blind, placebocontrolled, randomized study”, J. Urol., Vol. 181, No, 1, 2009, pp.741-751, Epub December 2008, discloses the use of NAC and Se incombination or separately for improving semen quality in infertile men.Administering NAC and Se in combination resulted in additive beneficialeffects.

Yalçin et al., “Synergistic action of sodium selenite andN-acetylcysteine in acetaminophen-induced liver damage”, Hum. Exp.Toxicol., Vol. 27, No. 5, 2008, pp. 425-429, discloses the use of NACand Se in combination or separately for treatment of acetaminophenoverdosing. NAC and Se in combination were found to give betterprotection against hepatotoxicity compared to either agent alone.

Emonet et al., “Thiols and selenium: protective effect on human skinfibroblasts exposed to UVA radiation”, J. Photochem. Photobiol., Vol.40, No. 1, 1997, pp. 84-90, discloses the use of NAC and Se incombination to protect cells against UVA damage.

Look et al., “Sodium selenite and N-acetylcysteine inantiretroviral-naive HIV-1-infected patients: a randomized, controlledpilot study”, J. Clin. Invest., Vol. 28, No. 5, 1998, pp. 389-397,discloses a combined oral administration of NAC and Se with theobjective to improve blood count and reduce viral load in patients withHIV.

Sener et al., “Melatonin and N-acetylcysteine have beneficial effectsduring hepatic ischemia and reperfusion”, Life Sciences, Vol. 72, 2003,pp. 2707-2718, discloses the use of NAC and Mel alone or in combinationto treat hepatic ischemia. Mel was found more potent than NAC, and thecombination of the two was found to be more effective than either alone.

WO 03/077900 A1 and US 2005/0164911 A1 disclose a method for preventingthe development of cancer or neurodegenerative diseases by administeringNAC, melatonin, or a combination thereof, as well as a medicamentcomprising NAC and Mel.

WO 00/531376 describes a composition containing cysteine, selenium andmelatonin together with a number of other components. US 2007/0231312,US 2011/027771, WO 98/33494 and U.S. Pat. No. 6,207,190 all describedifferent formulations containing among other things, NAC, Se and Mel.Further, US 2004/045566 describes a composition containing glutathione,selenium and melatonin for absorbing dangerous component from tobaccosmoke. None of the said documents describes the use of selenomethionine.

In all documents referred to in the prior art selenium is used as such.

SUMMARY

The present disclosure provides a solution to the problem of increasingthe efficacy of NAC. In one embodiment, a medical product orpharmaceutical combination providing increased efficacy ofN-acetyl-L-cysteine (NAC) is provided. In another embodiment, a medicalproduct or pharmaceutical combination providing increased efficacy ofNAC for use in the treatment of diseases in mammals, such as humans,e.g. benign and malignant iperproliferations including various types ofcancers, autoimmune diseases, neurodegenerative diseases,endocrinological diseases, type 2 diabetes, all types of fibrosis,amyloidosis, endometriosis, polycystic ovary syndrome, dysmenorrhea,dermatological diseases including vitiligo, alopecia and psoriasis andbacterial infections, is provided. In yet another embodiment, a medicalproduct providing increased efficacy of NAC for use as an antibacterialagent and for cosmetic purposes is provided.

The present disclosure shows that the effect of N-acetyl-L-cysteine(NAC) is enhanced when NAC is administered together with selenium (Se),in the form of selenomethionine, and melatonin (Mel). Selenomethionineand Mel in combination strongly increase NAC efficacy and also allow areduction in NAC concentration, thus ameliorating its efficacy in bothshort term and prolonged treatments. After testing in vitro and in vivothe efficacy of this combinatorial treatment, with NAC, Se (e.g. in theform of selenomethionine, SeMet in the following) and Mel, it was shownthat undesirable effects of Se and Mel are avoided.

Thus the present disclosure provides a combination of NAC, SeMet and Meluseful for the treatment of a variety of diseases and conditions, e.g.benign and malignant iperproliferations including various types ofcancers, autoimmune diseases, neurodegenerative diseases,endocrinological diseases, diabetes, all types of fibrosis, amyloidosis,endometriosis, polycystic ovary syndrome, dysmenorrhea, dermatologicaldiseases and bacterial infections. The combination is also useful as anantibacterial agent and for cosmetic purposes. In addition, the presentdisclosure provides a medical product and a pharmaceutical compositioncomprising said combination, as well as a method of treatment comprisingsimultaneous administration of NAC, SeMet and Mel to a patient.

In one aspect, the present disclosure provides a medical productcomprising, separately or together, N-acetyl-L-cysteine,selenomethionine and melatonin, and/or physiologically acceptable saltsthereof. By “separately” is meant that the three substances are part ofthe medical product, but may be provided in separate compartments, asseparate units, of the medical product. By “together” is meant that thethree substances may alternatively be provided in the same part of themedical device, e.g. as a mixture. The medical product is in one aspectof the disclosure used for simultaneous, sequential or separatecombinatorial administration to a mammal.

In one embodiment, the components of the medical product are for oraladministration; e.g. N-acetyl-L-cysteine for administration at a dose of5-45 mg/kg/day, selenium, in the form of selenomethionine, foradministration at a dose of 0.4-1.2 μg/kg/day and melatonin foradministration at a dose of 0.02-0.08 mg/kg/day. The medical product isin one embodiment a pharmaceutical composition comprisingN-acetyl-L-cysteine, selenium in the form of selenomethionine, andmelatonin.

The medical product comprising, separately or together,N-acetyl-L-cysteine, selenium in the form of selenomethionine, andmelatonin is in another embodiment for epicutaneous administration. Insuch medical product, N-acetyl-L-cysteine is in one embodiment foradministration at a concentration of 3-10 wt-%, selenium in the form ofselenomethionine, is for administration at a concentration of 0.3-1 wt-%and melatonin is for administration at a concentration of 0.01-0.2 wt-%.In one embodiment, the medical product is a pharmaceutical compositioncomprising N-acetyl-L-cysteine, selenium, in the form ofselenomethionine, and melatonin.

In one embodiment, the medical product is for treatment of benign ormalignant neoplasies, including various types of cancers. In anotherembodiment, it is for cosmetic treatment of skin and for treatment ofdermatological diseases. The medical product may also be used for thetreatment of autoimmune diseases, neurodegenerative diseases,endocrinological diseases, type 2 diabetes, all types of fibrosis,amyloidosis, endometriosis, polycystic ovary syndrome, dysmenorrhea andother diseases. In one embodiment, the medical product is for use as anantibacterial agent.

The medical product, comprising N-acetyl-L-cysteine, selenium in theform of selenomethionine and melatonin, is in another aspect of thedisclosure for use as an antibacterial agent, for use in, together with,or on the surface of a medical device that is adapted for use in contactwith body fluids. The medical product may for instance be used in acoating for such a medical device, or may be incorporated into thematerial, e.g. plastics, of such a medical device. In one embodiment ofthe disclosure, N-acetyl-L-cysteine, selenium in the form ofselenomethionine, and melatonin are comprised in a hydrogel coating fora medical device. The medical product for use as an antibacterial agentmay further comprise additional active compounds such as germicidaldrugs, noble metal solutions and/or dexamethasone.

In one aspect, the present disclosure provides an antibacterial agentcomprising, separately or together, N-acetyl-L-cysteine, selenium in theform of selenomethionine, and melatonin, and/or physiologicallyacceptable salts thereof. The antibacterial agent is in one embodimentfor use in a hydrogel coating for a medical device. In anotherembodiment, it is for integration with the plastics of a medical device.

In another aspect, the present disclosure provides a medical devicecomprising a medical product comprising N-acetyl-L-cysteine, selenium inthe form of selenomethionine, and melatonin, and/or physiologicallyacceptable salts thereof and optionally an additional active compoundsuch as germicidal drugs, noble metal solutions and/or dexamethasone.

In one aspect, the present disclosure provides use of a medical productcomprising, separately or together, N-acetyl-L-cysteine, selenium in theform of selenomethionine and melatonin, and/or physiologicallyacceptable salts thereof, for the treatment of benign and malignantneoplasia including various types of cancers, autoimmune diseases,neurodegenerative diseases, endocrinological diseases, type 2 diabetes,all types of fibrosis, amyloidosis, endometriosis, polycystic ovarysyndrome, dysmenorrhea, dermatological diseases including vitiligo,alopecia or psoriasis or bacterial infections. The disclosure alsoprovides use of the medical product for cosmetic treatment of skin andas an antibacterial agent with a medical device that is adapted for usein contact with body fluids.

In one aspect, the present disclosure provides a method of treatment ofa disease, or for cosmetic purposes, comprising simultaneous, sequentialor separate combinatorial administration of N-acetyl-L-cysteine,selenium in the form of selenomethionine and melatonin, and/orphysiologically acceptable salts thereof, to a human or mammal patient.The disease or cosmetic purpose is selected from benign and malignantneoplasia including various types of cancers, autoimmune diseases,neurodegenerative diseases, endocrinological diseases, type 2 diabetes,all types of fibrosis, amyloidosis, endometriosis, polycystic ovarysyndrome, dysmenorrhea, dermatological diseases including vitiligo,alopecia or psoriasis or bacterial infections, or cosmetic treatment ofskin.

BRIEF DESCRIPTION OF THE FIGURES

Embodiment of the present invention will be explained in more detail inthe following description, referring to the enclosed figures, where:

FIG. 1 shows the metabolic recycling of glutathione (GSH) byselenium-dependent glutathione peroxidase (cGPx(Se)) and glutathionereductase (GR). The activity of GR requires NADPH which is supplied bythe activity of the enzyme glucose-6-phosphate dehydrogenase (G6PDH).G6P: glucose-6-phosphate; 6PG: 6-phosphogluconate; RO.: alkoxyl radical;.OH: hydroxyl radical; ROOH: hydroperoxide.

FIG. 2 shows the effect of a three months treatment with NAC+SeMet+Melon a patient with vitiligo, as described in Example 1.

FIG. 3A shows the effect on skin texture of a one month treatment withNAC+SeMet+Mel, as described in Example 2. Microscopy images of siliconrubber skin replicas showing a network of thin lines characterizing themicrorelief of skin surface. Note the more regular network aftertreatment. Magnification: 5× or 10×, as reported in the panels.

FIG. 3B shows the Fast Fourier transform (FFT) of some of the images inFIG. 3A showing an irregular pattern prevalent in a single direction inthe samples before treatment (A and B), while a regular circular patternis observed after treatment (C and D).

FIG. 4 shows results from experiments described in Example 3. K562 cellswere treated with 0.2 mM menadione and/or 0.4 mM NAC and/or 0.2 mM SeMetand/or 2 mM Mel as indicated. In the histogram, the indication Se alwaysrefers to SeMet.

FIG. 5 shows results from experiments described in Example 3. HL60 cellswere treated with 0.133 mM menadione and/or 0.4 mM NAC and/or 0.2 mMSeMet and/or 2 mM Mel as indicated. In the histogram, the indication Sealways refers to SeMet.

FIG. 6 shows results from experiments described in Example 3. HL60 cellswere treated with 0.5 mM menadione and/or 1 mM NAC and/or 0.2 mM SeMetand/or 1 mM Mel as indicated. In the histogram, the indication Se alwaysrefers to SeMet.

FIG. 7 shows results from experiments described in Example 3. HL60 cellswere treated with 0.133 mM menadione, 0.2 mM SeMet, 2 mM Mel anddifferent NAC concentrations as indicated.

FIG. 8 shows results from experiments described in Example 4,demonstrating the antiproliferative and differentiating action of NAC incombination with SeMet and Mel. Average fold changes of the inhibitor ofproliferation gene P21 (average of 5 exps.) and of the differentiationmarkers SI (5 exps.), ALPI (4 exps.), CLDN1 (6 exps.) and PCDH1 (5exps.). Significance, p<0.05, excluding two samples marked with (*)where the combination NAC+Se and NAC+Mel were not significantlydifferent from that of NAC+SeMet+Mel. In the histogram, the indicationSe always refers to SeMet.

FIG. 9 shows a comparison of the effect of NAC alone with thecombination of the three substances. In the histogram, the indication Sealways refers to SeMet.

DETAILED DESCRIPTION

NAC

N-acetyl-L-cysteine (NAC) is a well-known low molecular weightpharmaceutical drug, with the chemical formula

For the purpose of the present invention NAC may also be administered inits dimeric form (di-NAC).

NAC has been found to exert molecular and physiological effects throughvarious mechanisms. The features of NAC are mainly related to its thiolgroup, which makes it effective in most biochemical pathways were theglutathione (GSH) acts. NAC is processed by cells to L-cysteine and isused in the de novo synthesis of GSH, thus being considered a precursorof GSH.

Although details of NAC mechanisms of action are not completelyunderstood, undoubtedly they have to be attributed to its thiol group.It is thus involved in the complex redox cycling of thiol groups in thecell, and thereby affects the regulation of the redox state of the cellas well as intracellular and intercellular signaling. In this redoxcycling several enzymes act whose redox transitions occur via theoxidation/reduction of glutathione (GSH). There is previous evidence ofNAC involvement in this picture, as summarized in FIG. 1, NAC having asimilar or even higher efficiency as compared to GSH. In this respect,NAC can be considered a potent antioxidant.

Of extreme physiological importance is the disulfide formation andbreakage cycle, a common mechanism by which protein activity andcellular signaling is regulated. Enzymes such as protein tyrosinephosphatases and tyrosine kinases, for example, play pivotal roles inthe control of the cell cycle, cell proliferation and differentiation,and many of them are regulated by the redox state of their cysteines.

NAC has been and still is largely used as a mucolytic agent, where themode of action is generally attributed to the redox breakage ofsensitive cysteine disulfur bridges in the mucus proteins. In additionNAC has been the prime treatment of paracetamol poisoning.

In recent years, NAC has also been acknowledged as having otherbeneficial properties. NAC has for instance been reported to have ananti-inflammatory effect, a reason for its addition to the family ofnon-steroidal anti-inflammatory drugs (NSAIDs). NAC has further beenfound to inhibit cell proliferation and promote quiescence, furtherevolving in terminal differentiation. NAC has in this context been foundto possess a marked antiproliferative effect on cancer cells and hasalso been found to be effective in the treatment of endometriosis. Forexample, the present inventors relatively recently found that NACpossesses a marked antiproliferative effect on cancer cells ofepithelial origin (Cell Death and Differentiation 2005, 12(10):1285-1296; BMC Cancer 2005, 5: 75).

The antiproliferative effect of NAC, in the study of cancer, was notrelated to cell death or to toxicity but, instead, was due to theactivation of a physiological differentiation pathway, which can beregarded as a normalization of cell functions towards the tissue oforigin.

A relevant advantage in the use of NAC is the virtual absence of sideeffects. A disadvantage is its decreased efficacy in prolongedtreatments, with a reported decline in plasma levels so that therequired dose increases, thereby also increasing the risk of someundesired effects.

NAC in Combination with Se and Mel

On this basis, the present inventors aimed at a reduction in theeffective NAC dose by the contemporary use of other drugs that can exerta synergism with NAC. A reduction in NAC effective dose is useful inboth long-term and short-term treatments. For that purpose the attentionwas focused to the enzyme network governing thiol redox cycling,particularly those reactions involving glutathione recycling by means ofenzymes such as glutathione peroxidase (GPx) and glutathione reductase(GR). The focus on the flow of these redox reactions is justified by theobservation that they account for nearly 99.9% of redox reactions inbiology (DP Jones. Am. J. Physiol. Cell Physiol. 2008; 295:C849-C868)and that NAC action should be mostly related to a modification of theactivity of these multiple enzymes (Parasassi T, Brunelli R, Costa G, etal. TheScientificWorldJOURNAL 2010; 10:1192-1202).

A decrease in the expression of GR after prolonged treatments with NAChas been reported (L Pendyala and P J Creaven, Cancer EpidemiolBiomarkers Prev 1995; 4:245-251). Reports also exist showing that Melsupplementation may yield an increased expression and/or activity of GRand also of another relevant enzyme in thiol redox reaction: theglutathione peroxidase (GPx) (Hardeland R, Cardinali D P, Srinivasan V,et al. Prog Neurobiol. 2011; 93(3):350-84, Epub Dec. 28, 2010; García T,Esparza J L, Giralt M, et al., Biol Trace Elem Res. 2010; 135:220-32).

In this picture, Se represents a cofactor of several glutathioneperoxidases (GPx). Se is necessary GPx activity (see for instance Bulatoet al. Free Radic Biol Med. 2007; 42:118-23), and does not exert anyspecific antioxidant action in the absence of the GPx enzymes.

The present inventors therefore reasoned that to ensure a balancedaction, devoid of undesired effects, Se had to be in the form of SeMetand administered together with Mel, i.e. in conditions where GR and GPxare stimulated and GPx can fully exert its activity. In this context,NAC could warrant the reduction potential for the overall kinetics toproceed—in this, eventually being more effective than glutathione (GSH).

On the basis of the biochemistry of the thiol redox system, the presentinventors aimed at increasing the effect of NAC, at reducing thetherapeutic concentration of NAC and to assure NAC efficacy in prolongedtreatments. The purpose was achieved by stimulation of elements presentin the thiol redox system.

With the aim of: 1) restoring/enhancing the expression/activity ofenzymes in charge of the thiol signaling redox control; 2)reestablishing the proper NAC action even in prolonged treatments; and3) reducing the effective NAC concentration, NAC treatment was combinedwith selenium in the form of selenomethionine and melatoninsupplementation. This combination was inspired by the requirement ofselenium for the activity of GPx, and by the reported effect ofmelatonin in increasing the expression/activity of GR and possibly ofGPx.

Thus, the present disclosure is based on the purpose of enhancing NACaction through enhancement of the thiol redox system by:

-   -   1) stimulating the activity of glutathione peroxidase (GPx) by        Se supplementation, in the form of selenomethionine (SeMet),        which is a bioavailable form of Se.    -   2) The use of selenomethionine instead of selenium is very        important for the effect. [2) increasing the expression and/or        activity of glutathione reductase (GR) and glutathione        peroxidase (GPx) by Mel.

NAC action in combination with these two additional substances wasverified by testing the antioxidant, antiproliferative anddifferentiating effects of NAC. The combinatorial effect and possiblesynergy between NAC, Mel and Se in the form of SeMet was tested in vitroby using two cell lines challenged with an oxidative stimulus (Examples3 and 4), as well as in vivo in a patient with vitiligo and in adermatological study (Examples 1 and 2).

With regard to the antioxidant effect of NAC in the thiol redox system,measured as a decrease in the stimulated production of hydrogenperoxide, it was found that:

1) NAC action is definitely enhanced by this association. In severalcases NAC was almost three times more effective in combination withSeMet and Mel than by itself.

2) NAC concentration could be decreased, a similar effect being reachedusing less than half of the concentration.

3) Surprisingly, in some cellular systems, an unfavorable oxidant effectwas observed when combining only two of the substances, i.e. NAC+SeMetor of NAC+Mel. This is surprising since these combinations are reportedas beneficial in the scientific literature (Safarinejad M R. J Urol.2009; 181:741-51. Yalçin S et al. Hum Exp Toxicol. 2008; 27:425-9). Incontrast, it was observed that the combination of the three substancesrestored a cell protection, with a final effect of an almost completeabsence of oxidative effect.

With regard to the effect of NAC relating to decreasing cellproliferation and inducing a differentiation process, it was found that:

1) the antiproliferative action of NAC is doubled by the combinationwith SeMet and Mel as compared to use of NAC by itself.

2) the differentiating action of NAC was definitely enhanced by theabove combination, being 30-50% higher as compared to use of NAC byitself, depending on the specific marker.

3) by analyzing some specific differentiation markers, it was observedthat the combinations of NAC+SeMet or NAC+Mel had an effect comparableto the combination of the three substances. Nevertheless, there is aclear trend to an overall increased NAC action when the three substancesare used in combination.

Administration

The novel combination described herein, of NAC, Se, in the form of SeMetand Mel, may be administered to a mammal, such as a human,simultaneously, sequentially or separately in combination. By“simultaneous combinatorial administration” is meant that all threesubstances are administered together to the mammal at the same point intime. By “sequential combinatorial administration” is meant that thethree substances are administered to the mammal during the same periodof treatment, e.g. during the same day of treatment, week(s) oftreatment or month(s) of treatment, such that all substances are presentin the mammal in combination, the substances being administered, i.e.given to the mammal at different time points in a set order. By“separate combinatorial administration” is meant that the threesubstances are likewise administered to the mammal during the sameperiod of treatment such that all substances are present in the mammalin combination, but the substances being administered, i.e. given to themammal at different time points in an irregular order administration maybe topical or systemic (either enteral or parenteral). Typical ways ofadministration include oral administration and epicutaneousadministration. Other possible ways of administration includeintravenous, intradermal, subcutaneous, intramuscular, intravaginal,intrauterine, intraperitoneal, rectal, nasal, intrathecal, inhalationaland intravesical administration.

Formulations

The combination of NAC, SeMet and Mel according to the presentdisclosure may be administered as separate pharmaceuticalformulations/compositions, each comprising only one of the three activeingredients, i.e. NAC or SeMet or Mel respectively. Alternatively thecombination may be administered in a pharmaceutical compositioncomprising two of or all three of the active ingredients. In eithercase, pharmaceutical compositions comprising either only one orcombinations of NAC and/or SeMet and/or Mel may be prepared in a mannerper se known by a person skilled in the pharmaceutical art.

The pharmaceutical composition may for instance be adapted for oraladministration. Such compositions could be administered in differentforms, such as tablets. Other forms, such as capsules, suppositories,solutions, suspensions, syrups or the like are also conceivable. Inpharmaceutical formulations in the form of dosage units for oraladministration, the active ingredient or ingredients may be mixed with asolid, powdered carrier or excipient that serves as a vehicle or mediumfor the active ingredient, such as lactose, saccharose, sorbitol,mannitol, starch, amylopectin, cellulose derivatives, gelatin, citricacid, sodium citrate, sodium (acid) carbonate or another suitablecarrier; stabilizing substances such as alkaline compounds e.g.carbonates, hydroxides and oxides of sodium, potassium, calcium,magnesium and the like as well as with lubricating agents such asmagnesium stearate, calcium stearate, sodium stearyl fumarate andpolyethylene glycol waxes. The mixture is then processed into granulesor pressed into tablets. Granules and tablets may be coated with anenteric coating which protects the active compound from acid catalyzeddegradation as long as the dosage form remains in the stomach. Theenteric coating is chosen among pharmaceutically acceptable entericcoating materials e.g. beeswax, shellac or anionic film-forming polymersand the like, and in some embodiments, in combination with a suitableplasticizer. To the coating, various dyes may be added in order todistinguish among tablets or granules with different amounts of theactive compound present.

Soft gelatin capsules may be prepared with capsules containing a mixtureof the active compound, vegetable oil, fat or other suitable vehicle forsoft gelatin capsules. Soft gelatin capsules may also be enteric coatedas described above.

Hard gelatin capsules may contain granules or enteric coated granules ofthe active compound. Hard gelatin capsules may also contain the activecompound in combination with a solid powdered carrier such as lactose,saccharose, sorbitol, mannitol, potato starch, amylopectin, cellulosederivatives or gelatin. The capsules may be enteric coated as describedabove.

One option is to provide NAC and/or SeMet and/or Mel in a slow-releaseformulation (also denoted sustained-release or controlled-release). Bybeing able to reduce the rate of diffusion and uptake of activesubstances into the blood stream such a formulation enablesadministration of a larger dose at longer intervals. The dose is thendistributed in the blood over a long time in small quantities, e.g. over12+12 hours in the case of a twice-a-day regimen scheme. Many differenttechnologies and formulations for slow-release are since long known inthe art and may be applied with the present disclosure. In suchtechnologies the active substance is for example encapsulated in acoating or matrix that is insoluble or less soluble in the body fluidwhere it is administered. Formulations having a combined effect ofslow-release and gastric protection is also possible and may be usedwithin the present disclosure.

Liquid preparation for oral administration may be prepared in the formof syrups or suspensions, e.g. solutions or suspensions containing from0.2% to 20% by weight of the active ingredients and the remainderconsisting of sugar or sugar alcohols and a mixture of ethanol, water,glycerol, propylene glycol and/or polyethylene glycol. If desired, suchliquid preparations may contain colouring agents, flavouring agents,saccharine and carboxymethyl cellulose or other thickening agents.Liquid preparations for oral administration may also be prepared in theform of dry powder to be reconstituted with a suitable solvent prior touse.

For topical administration, NAC and/or SeMet and/or Mel may be appliedin pure form, i.e. as liquids. In exemplary embodiments, they areadministered in compositions/formulations comprising a dermatologicallyacceptable carrier, which may be a solid or a liquid, i.e. as anemulsified cream, ointment, lotion, liniment, powder or the like.Examples of liquid carriers include water, alcohols or glycols orwater-alcohol/glycol blends, in which the present compounds can bedissolved or dispersed at effective levels, optionally with the aid ofnon-toxic surfactants. Thickeners such as synthetic polymers, fattyacids, fatty acid salts and esters, fatty alcohols, modified cellulosesor modified mineral materials may be added to form ointments, lotions,pastes, gels and the like. Examples of solid carriers include finelydivided solids such as talc, clay, microcrystalline cellulose, silica,alumina and the like. Adjuvants such as fragrances and additionalantimicrobial agents may also be added. In an exemplary embodiment, agalenic formulation for topical administration comprises Aqua, Ceteareth25 Glyceryl Stearate, Tea Stearate, Cetearyl alcool, Caprylic/CapricTriglyceride, Dimethicone, Glyceryn Hydrogenated Polyisobutene,Polysorbate 60, Pentylene Glycol and optionally vanilla.

For intravenous or intraperitoneal administration, solutions of theactive compounds can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils, optionally encapsulated in liposomes. The ultimate dosage formshould be sterile, fluid and stable under the conditions of manufactureand storage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), vegetable oils, nontoxic glyceryl esters, and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe formation of liposomes, by the maintenance of the required particlesize in the case of dispersions or by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manyembodiments, isotonic agents, for example, sugars, buffers or sodiumchloride, are included. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin. Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, in some embodiments, themethods of preparation are vacuum drying and the freeze dryingtechniques, which yield a powder of the active ingredient plus anyadditional desired ingredient present in the previously sterile filteredsolutions.

Dosage units for rectal administration may be prepared in the form ofsuppositories which contain the active substances mixed with a neutralfat base, or they may be prepared in the form of a gelatin rectalcapsule which contains the active substances in a mixture with avegetable oil, paraffin oil or other suitable vehicle for gelatin rectalcapsules, or they may be prepared in the form of a ready-made microenema, or they may be prepared in the form of a dry micro enemaformulation to be reconstituted in a suitable solvent just prior toadministration.

Solutions for parenteral administration may be prepared as solutions ofthe active ingredients of the present disclosure in pharmaceuticallyacceptable solvents, such as in a concentration from 0.1 to 10% byweight. These solutions may also contain stabilizing agents and/orbuffering agents and may be manufactured in different unit dose ampoulesor vials. Solutions for parenteral administration may also be preparedas dry preparations to be reconstituted with a suitable solventextemporaneously before use.

With regard to NAC, the present disclosure typically requires a strictassessment of the pharmaceutical quality of NAC preparation forobtaining the effective dose. NAC is not a stable molecule, its activethiol residue can be easily oxidized by oxygen, light and otherradiations, so that the effective dose would not be reached. In variousembodiments, the preparation is thus protected from light. For oraladministration, NAC can be prepared in soluble tablets with sodiumhydrogen carbonate, which helps in a partial removal of oxygen fromwater during dissolution.

It has been observed that high doses of NAC may cause abdominal pain.For overcoming this, an option is to provide NAC in a formulation withgastric protection, suitable for preventing NAC release/solubility inthe stomach. Such formulations are well known in the art and may be usedwith the present disclosure. For example, tablet coatings that areresistant to gastric fluids and allow release of the drug only in theintestine, after its transit through the stomach, may be used. Commonlyused formulations include polymers such as cellulose derivatives ormethacrylate amino ester copolymers. The coating protects the tabletcore from disintegration in the acidic environment of the stomach byemploying pH sensitive polymer, which swell or solubilise after havingpassed through the stomach, in response to an increase in pH, to releasethe drug.

Dosages

For oral administration to a human, as well as other mammals, a suitabledaily dose of NAC is approximately 5 and 45 mg/kg/day, such as 20-30mg/kg/day. A suitable daily dose of SeMet is approximately 0.0004-0.0012mg/kg/day, such as approximately 0.0008 mg/kg/day, and a suitable dailydose of Mel is approximately 0.02-0.08 mg/kg/day, such as approximately0.04 mg/kg/day. The dose may be given once a day or may be divided intwo or more, for example three or four, daily administrations of eitherone or two doses (e.g. pills) each, where each dose may comprise e.g.0.15-3.0 g of NAC, for example 0.3-0.6 g of NAC and for example threetimes a day; 0.028-0.110 mg of SeMet such as approximately 0.055 mg ofSeMet; and 1-6 mg of Mel such as approximately 3 mg of Mel.

For oral administration, the daily dosage weight ratio of NAC to SeMetis within the range of 4000:1 to 60 000:1, for example within the rangeof 5000:1 to 40 000:1 or 25 000:1 to 37 000:1, such as 32 500:1. Thedaily dosage weight ratio of NAC to Mel is within the range of 60:1 to2000:1, for example 150:1 to 1000:1 or 500:1 to 750:1, such as 600:1.Typically the three substances are administered at a daily dosage weightratio of NAC to SeMet to Mel that is approximately 32 500:1:54. Thesubstances may be administered simultaneously, separately or in acomposition comprising all three substances, in said ratios.

For epicutaneous administration (e.g. by emulsion cream, ointments,lotions, liniments or similar) a suitable concentration of NAC isapproximately 3-10 wt-%, for example 5 wt-%. A suitable concentration ofSeMet is approximately 0.3-1 wt-%, for example 0.5 wt-%, and a suitabledaily dose of Mel is approximately 0.01-0.2 wt-%, for example 0.1 wt-%.The cream, lotion or similar may be applied once a day or may be appliedin two or more, such as three or four, daily administrations.

For epicutaneous administration, the weight-% ratio of NAC to SeMet iswithin the range of 3:1 to 33:1, for example approximately 10:1. Theweight-% ratio of NAC to Mel is within the range of 15:1 to 1000:1, forexample approximately 50:1. Typically, the three substances areepicutaneously administered at a weight-% ratio of NAC to SeMet to Melthat is approximately 50:1:5. The substances may be administeredsimultaneously, separately or in a composition comprising all threesubstances, in said ratios.

Periods of treatment may last for one or a few days up to severalmonths, depending on the disease or condition to be treated.

For longer periods of treatment with NAC and SeMet and Mel, e.g. 1-2months or more, the treatment may be intermittent. By “intermittentadministration or treatment” is meant that the treatment is interruptedin periods, i.e. that the pharmaceutical composition is administered fora period of time, e.g. a few days, followed by an interruption inadministration, where no pharmaceutical composition is administered fora period of time, e.g. for a few days, and then followed byadministration again. Intermittent treatment can be regular, e.g.treatment for a fixed number of days or weeks, followed by interruptionfor a fixed number of days or weeks. Examples include repeated schemeswith treatment for 4 days followed by interruption for 3 days each weekor treatment for 2 weeks followed by interruption 1 week. A special caseof regular intermittent treatment is pulsed treatment, i.e. with regulartreatment and interruption duration, e.g. administration every other dayor administration for two days followed by two days of interruption etc.Irregular intermittent treatment schemes that are not regularly repeatedor have a more complex scheme that is repeated is also conceivable, e.g.dependent on response to treatment. In different exemplifyingembodiments of the present disclosure, the prescribed dose of NAC andSeMet and Mel is administered for 3-5 consecutive days followed by 2-4days of interruption, or administered for 1-3 consecutive days followedby 1-2 days of interruption.

The dose of NAC, SeMet and Mel will depend on the particular formulationselected, the route of administration, the nature of the disease orcondition to be treated as well as the weight, age, condition andspecies (human or animal) of the patient.

A known dose of NAC to treat a particular disease or condition maytypically be approximately halved when NAC is administered together withthe optimal dosages of SeMet and Mel. As an example, NAC for thetreatment of endometriosis is in an embodiment, administered alone at adose of 20-90 mg/kg/day, such as 30-60 mg/kg/day, orally, for two monthsor more. When NAC is administered in combination with SeMet and Mel theNAC dosage can be lowered to 15-30 mg/kg/day for two months or more.

Use/Medical Indications of the Present Disclosure

The present disclosure, comprising a combination of NAC, Se asselenomethionine and Mel, may be used for any purpose where NAC may beused or is known to have an effect, e.g. in diseases where glutathioneis involved and/or in which thiol (—SH) reduction/oxidation (redox)switches play a fundamental role, as in the case of signaling, alsodriven by hormones, or where NAC's antiproliferative andprodifferentiating effect, antioxidant or antiinflammatory effects maybe utilized. It may for example be used for the treatment of benign andmalignant neoplasia, including various types of cancers, type 2diabetes, various types of fibrosis, endometriosis, polycystic ovarysyndrome, dysmenorrhea and dermatological diseases, including vitiligoand psoriasis. Due to the relevance of oxidation phenomena in proteinaggregation to form amyloids, the present disclosure can be used for theprevention and treatment of various types of amyloidosis, such asAlzheimer's disease. In addition, it may be used as an antibacterialagent and for cosmetic purposes. Other uses also include treatment ofHIV/AIDS, chemical and infectious hepatitis, cataracts, Parkinson'sdisease, chronic obstructive pulmonary disease, asthma, radiationpoisoning, malnutritive states, arduous physical stress, aging, sepsis,trauma, burns, bipolar disorder, major depressive disorder, andschizophrenia, and for patients with suboptimal immune response.

As an example of treatment of dermatological diseases, one patient withvitiligo was treated with the combination of the three substances andafter three months of treatment an extreme reduction of depigmentationwas observed (Example 1, FIG. 2).

As an example of cosmetic treatment, skin texture on a subject whoseface skin was treated for one month with a galenic preparationcontaining the three compounds, was monitored. After one month ofcombined treatment with a cream preparation containing NAC, SeMet andMel, face surface lines appeared more regular, the skin appeared morecompact and smoother and skin irregularities and pimples were reduced.

Combination of NAC, SeMet and Mel for Use as an Antibacterial Agent

The present inventors also show (Example 5) that the combination of NAC,SeMet and Mel is effective for use as an antibacterial agent, e.g. whencomprised in a coating on medical instruments and devices such ascatheters of latex or polyvinyl chloride. Alternatively, the combinationof NAC, SeMet and Mel may be incorporated in plastic materials for usein medical devices.

For instance, hydrogel coating may be used as a method for solidsubstrate surface modification. Examples of hydrogel coatings includewater insoluble hydrogel based on polyurethane (PUR) orpolyvinylpyrrolidone (PVP). Besides advantages like improving materialbiocompatibility, hydrophilization and lubrication, hydrogel coatingbrings the additional possibility of incorporating active agents to thesurface, e.g. NAC, SeMet and Mel. Combinations of a hydrophilic matrixand a hydrophobic drug composition of NAC, SeMet and Mel seem to beespecially promising.

Hydrogel modified surfaces have the additional advantage over unmodifiedsurfaces, in that they can serve as a drug reservoirs for a local drugdelivery. For example, in certain circumstances drug dosage time shouldlast at least a few hours, but no longer than 3 days. It can bedesirable e.g. in case of implantation of devices like tracheotomytubes, when anti-inflammatory active substances should be released atthe very beginning, to prevent later side effects like trachealstenosis, but later should not interrupt normal cell divisions in thesubsequent healing process and epithelium formation. By using ahydrophilic hydrogel matrix with hydrophobic NAC, SeMet and Mel, thisrelease profile can easily be obtained due to the properties of thehydrophilic matrix as controlled drug release system during itsswelling.

In addition to NAC, SeMet and Mel, further active substances may beadded to coatings or plastics for use with medical instruments anddevices, e.g. germicidal drugs, noble metal solutions and/ordexamethasone.

The active agent, i.e. NAC, SeMet and Mel, and possibly one or moreadditional active substance(s) may be incorporated to the coating in twodifferent modes: I. as a component of the solution in any step of thecoating formation, e.g. during polymerization of the hydrogel coating,or II. through an impregnation bath. The later mode II. is in oneembodiment, made as an additional step after formation of a hydrogelcoating, but may also be done without such coating, directly to themedical device. Mode II. allows for modification of devices likesilicone catheters or polyglycolic acid resorbable sutures withoutpolymeric coating step or with the addition of the active substancessubsequent to the polymeric coating step.

The effects of active agent incorporation have been verified by thepresent inventors through drug dissolution tests to the phosphatebuffered saline (PBS) with 20% methanol or ethanol, extraction, and, incase of germicidal drug, by the inhibited growth zones method (data notshown). The dependency of the rate of drug dissolution and the loadcapacity, as well as coating stability on the process parameters wereinvestigated. The present inventors observed a spreading effect of NAC,SeMet and Mel during hydrogel swelling, as solid microparticles of thesubstances (NAC, SeMet, Mel) were precipitated out of the coating layerto the surrounding solution.

The present inventors show (Example 5) that the use of hydrogel coatingscomprising NAC, SeMet and Mel on catheter material (polyvinyl chloride,latex) significantly reduce the growth of bacteria as well as theformation of bacterial biofilms on such material, compared to bothnon-coated backbone material and hydrogel coated material without NAC,SeMet and Mel.

EXAMPLES Example 1: Treatment of Vitilgo

Vitiligo is an acquired pigmentation disorder in which there is a lossof skin melanocytes. As a result, white patches appear on the skin indifferent parts of the body. The prevalence of vitiligo varies in therange of 0.1-2% worldwide. Vitiligo can be psychologically devastatingfor the affected patient and can affect quality of life, self-esteem,marriage, and employment (Halder R M, Chappell J L. Semin Cutan MedSurg. 2009; 28(2):86-92.). The precise pathogenesis remains elusive,with proposed mechanisms falling under the rubrics of autoimmune,biochemical, oxidant-antioxidant, neural, and viral. Several studiessuggest that accumulation of free radicals toxic to melanocytes leads totheir destruction. Indeed, compared with control patients, the red cellsof vitiligo patients have lower levels of glutathione.

In the present example, we report the case of a 45 year old woman withdiffuse vitiligo around her neck and in both armpits. She was treatedfor three months using the following commercially available drugs, inthe form of tablets taken orally, with the protocol: 0.6 g NAC, threetimes/day; SeMet, 55 micrograms/once a day; Mel, 3 mg/once a day; allthe three drugs for the same three days/week followed by 4 days ofinterruption (no administration).

Pictures of her neck and both armpits were taken at the beginning and atthe end of the three months treatment.

No adverse side effects were reported.

From the pictures in FIG. 2, a clear reduction in depigmentation isevident, with an almost complete repigmentation in both armpits.

Example 2: Cosmetic Treatment of Skin

Ageing of the skin can be macroscopically recognized because ofwrinkles, loss of elasticity, sagging and thinning which take placegradually in the adult. Therefore, determination of skin surface textureis of particular importance in the field of dermatology as suchmeasurements can be used for skin diagnostics and evaluation oftherapeutic or cosmetic treatments.

Of increasing relevance is the impact of environmental pollution onskin, often degraded by polluting agents with the associated ageing anddysfunctions. Skin is directly exposed to pollution with an individualsurface evaluated of about 2 square meters, larger than any other humantissue. Mechanisms for damages due to pollution mainly resides in theinduction of oxidative stress and inflammatory response. Thesemechanisms drive us to the use of NAC, in combination with SeMet andMel, in a case of mildly aged, oily skin, with pimples.

A 43 year old woman visited a dermatologist for her oily skin,presenting redness and pimples. She was treated for one month with agalenic preparation containing: Aqua, Ceteareth 25 Glyceryl Stearate,Tea Stearate, Cetearyl alcohol, NAC 5%, SeMet 0.5%, Mel 0.1%,Caprylic/Capric Triglyceride, Dimethicone, Glyceryn HydrogenatedPolyisobutene, Polysorbate 60, Pentylene Glycol, and vanilla.

Skin texture was evaluated on silicon rubber skin replicas, prepared bythe dermatologist then observed by microscopy (FIG. 3A), following areported procedure (Setaro M, Sparavigna A. Irregularity skin index(ISI): a tool to evaluate skin surface texture. Skin Res Technol. 2001;7(3):159-63.). As illustrated by these authors, through the analysis ofsurface lines, microscopy of skin replicas shows the variation inregularity of skin surface texture. After one month of combinedtreatment with a cream preparation containing NAC, SeMet and Mel, theface surface lines appeared more regular, with an impressively increasedorder in their network, particularly when compared with the randomcoiled appearance observed before the treatment. The Fast Fouriertransform (FFT) analysis of the images, performed following the methodof Setaro and Sparavigna, indeed showed (FIG. 3B) a pattern with apreferential direction before the treatment—as indicated by the arrowsin the left panels A and B, indicating an irregular texture. After thetreatment, the FFT analysis showed instead an almost completelyregularly circular pattern, indicating a regular skin texture.

After the month of treatment, both the dermatologist and the patientreported a visual decrease in skin irregularities with a more compactand smoother appearance, a normalization of the sebaceous secretion, andthe disappearance of redness and pimples.

Example 3: Antioxidant Effect on In Vitro Cultured Blastoid Cell Lines

Methods

The human lymphoblastoid HL60 and the proerythoblastoid K562 cell lineswere used, grown at 37° C. in RPMI 1640 medium (Invitrogen, Gibco,Milano, Italy), supplemented with 10% fetal bovine serum, 5 μg/mlpenicillin, 5 μg/ml streptomycin, 2 mM glutamine, and routinelysubcultured three times a week. Cells were treated either with menadionealone (control), with NAC only, with NAC+Se-methionine (SeMet), withNAC+Mel, or with NAC+SeMet+Mel. In cases of SeMet supplementation, suchsupplementation was performed two days before experiments, by using aSeMet solution in buffer (concentrations as indicated in description ofFIGS. 4-7). In cases of Mel supplementation, such supplementation wasperformed 20 hours before experiments, by using a solution in ethanol(concentrations as indicated in description of FIGS. 4-7). Ethanolconcentration in growth medium was ≦1.3% and controls only with thissolvent were also grown in parallel.

The effect of NAC, Mel, SeMet and of a combination of these substancesin protecting the cells against an oxidative stimulus was detected byusing the fluorescent probe5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate,acetyl ester (CM-H2DCFDA) (Invitrogen, Molecular Probes). CM-H2DCFDA isa cell-permeant indicator for reactive oxygen species that is notfluorescent until removal of its acetate groups by intracellularesterases, and oxidation within the cell. The increase in hydroperoxides(mainly H₂O₂) within the cell is thus monitored by increase influorescence. Oxidative stimulus for the increase in H₂O₂ was performedby using menadione.

Cells were counted, and care was taken to keep the ratio between cellsand the fluorescent probe constant. Washed cells were resuspended in 2ml HBSS at a concentration of 10⁶/ml, then transferred to a cuvette andequilibrated at 37±0.1° C. in the fluorimeter cell holder for 15 min,under mild stirring. Two μl of a solution of CM-H2DCFDA in DMSO werethen added and the fluorescence intensity read versus time. After thefirst 200 sec of reading, a proper aliquot of NAC solution in phosphatebuffer (concentrations as indicated in description of FIGS. 4-7), and 10μl of a 0.1 M solution of menadione in ethanol were added, and thefluorescence intensity reading continued. During intensity readings,samples were subjected to continuous mild stirring.

The fluorescence intensity was continuously monitored for 20 min in a K2fluorimeter (ISS Inc. Champaign, Ill.) equipped with a xenon arc lamp,by using excitation at 495 nm and emission at 530 nm, with 8 nmbandpass.

Results

Representative results of the oxidative response in the cells after therespective treatment are shown in FIGS. 4, 5 and 6. Differentconcentrations of menadione, NAC, SeMet and Mel were tested and gavedifferent responses, also depending on the cell line. Nevertheless, thecombination of the three substances always resulted as the mosteffective in preventing the oxidation-driven increase in fluorescence.After the oxidative challenge with menadione, the DCFDA fluorescenceintensity increased. NAC addition resulted in a relevant reduction inthe kinetics of this intensity increase, as evaluated by its slope. Thepresence of either SeMet or Mel together with NAC had different effectsdepending on the cell type and on concentration.

Notably, in some cases it was observed that the couples NAC+SeMet orNAC+Mel, instead of decreasing the response, accelerated thefluorescence increase, thus suggesting a pro-oxidant effect. Arepresentative example is reported in FIG. 6. Nevertheless, even inthese cases the combination of the three components, NAC+SeMet+Mel,resulted in the maximum decrease in the slope, and thus of the oxidativeresponse, relative to the control.

The powerful antioxidant defense operated by the combination ofNAC+SeMet+Mel allowed a 60% reduction in NAC concentration. This isshown in FIG. 7 where the protection exerted by 1 mM NAC is comparableto that obtained by using only 0.4 mM NAC in combination with SeMet andMel.

CONCLUSIONS

The combination of NAC with SeMet and Mel definitely increases theantioxidant defense of NAC. The combination exerts a synergistic effect,not only with regard to NAC action but also with regard to the action ofeach of the three components alone. In relation to NAC alone, in severalcases the antioxidant effect was three times as effective when NAC wasused in combination with SeMet and Mel.

NAC concentration could be decreased, a similar effect being reachedusing less than half of the concentration.

Surprisingly, in some cases, an unfavorable pro-oxidant effect of SeMetor of Mel, also in combination with NAC, was observed. Although themechanisms are still debated, adverse pro-oxidant effects were reportedin the literature both for Se and for Mel. Nevertheless, in ourexperiments the combination of the three substances, wherein Se is inthe form of SeMet, restored an antioxidant cell protection, with a finaleffect of negligible oxidation even after the oxidant challenge.

Example 4: Differentiating Effect in Colon Carcinoma Cells

NAC was previously reported to modulate cell proliferation anddifferentiation, specifically to reduce proliferation by switching cellstowards a normal differentiation pathway. Reportedly, this action occursin normal cells as well as in cancer cells.

The expression of genes related to proliferation and differentiation ina colon carcinoma cell line (Caco-2) treated with NAC, in combinationwith SeMet or Mel or both, was analyzed as below. Results are shown inFIGS. 8 and 9.

Methods

The colon carcinoma cells Caco-2 (subclone TC7, Chantret et al., J. CellSci. 107:213-225; 1994) were routinely maintained in high glucose DMEM(Gibco, Invitrogen, Milan, Italy) containing 10% heat-inactivated fetalbovine serum, FBS (Gibco, Invitrogen). Cells were seeded at a density of3×10³/cm² on six-well plates. 24 h after seeding cells were treated withNAC (2 mM) and/or SeMet (25 nM) and/or Mel (25 μM). After 72 hours oftreatment, cells were analyzed for gene expression.

Expression of genes characteristic of enterocyte differentiation wasevaluated; two genes encoding for brush border hydrolases, theintestinal alkaline phosphatase (ALPI) and the sucrase-isomaltasealpha-glucosidase (SI); two genes encoding for proteins of cell-celladhesion complex, the tight junction claudin 1 (CLDN1) and the adherentjunction protocadherin 1 (PCDH1). The expression of an importantinhibitor of cell cycle, the cyclin-dependent kinase inhibitor 1A(CDKN1A/P21^(waf1/cip1)) as a marker of cell cycle arrest that normallyaccompanies Caco-2 cell differentiation was also analyzed.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used ashousekeeping gene.

Specific primer sequences were designed using the software Primer-BLASTavailable at: http://www.nebi.nlm.nih.gov and are reported in Table 1.

Total RNA was extracted in TRIzol Reagent (Invitrogen). One microgram oftotal RNA from each sample was treated with 1 U of DNAse I (Invitrogen)and reverse transcribed with 200 U of SuperScript III ReverseTranscriptase (Invitrogen), using 250 ng of random primers in 20 μl(final volume). Real-time quantitative RT-PCR was performed on ABI Prism7000 (Applied Biosystems, Monza, Italy) using the 5 Prime RealMasterMixSYBR ROX 2.5× (Eppendorf, Milan, Italy) in a 25 μl reaction volumecontaining 10 ng of cDNA and 0.6 μl of each primer (10 μM), according tothe following protocol: 1 cycle of 10 min at 95° C., 40 cycles of threesteps including 20 sec at 95° C., 40 sec at 60° C., and 45 sec at 68° C.All experiments were performed as duplicate in three or more separateassays. A negative control (no template) was run with each reaction toverify the absence of contaminations, and the specificity ofamplifications was confirmed by melting curve analysis. All data wereanalyzed as averages with standard error and significance was evaluatedthrough the p parameter using ANOVA. For the determination of relativetranscript abundance, the 2^(−ΔΔCT) method was used (Livak K J,Schmittgen T D. Methods 2001; 25: 402-408.).

TABLE 1 SEQ ID Target mRNA Primers NO: Sequence SI forward  1ACGATGGGGAGGACACTGGCT NM_001041.2 reverse  2 TCCAAGTTGCATCCAGCGGGT ALPIforward  3 GTATGTGTGGAACCGCACTG NM_001631.3 reverse  4CTGGTAAGCCACACCCTCAT CLDN1 forward  5 TGGCTGTCATTGGGGGTGCG NM_021101.3reverse  6 GCAGCAGCCCAGCCAGTGAA PCDH1 forward  7 CCTATCCAGCCTGAGCTTTGNM_002587.3 reverse  8 GGCAGTGATATAGGGTGCGT P21/cdkn1a forward  9TGAGCTGCGCCAGCTGAGGT NM_000389.3 reverse 10 GCTGCTCGCTGTCCACTGGG GAPDHforward 11 GTCAGTGGTGGACCTGACC NM_002046.3 reverse 12AGGGGTCTACATGGCAACTGResults

Results are shown in FIGS. 8 and 9, showing the increase (fold change)in gene expression of the five genes, when cells are subjected to therespective treatment. The real-time quantitative RT-PCR data undoubtedlyshow that:

1) the antiproliferative action of NAC (as indicated by an increasedexpression of the P21 gene) is doubled by the combination with SeMet andMel, as compared to NAC alone.

2) the differentiating action of NAC (as indicated by an increasedexpression of the marker genes SI, ALPI, CLDN1, PCDH1) is definitelyenhanced by the above combination, with marker genes expression being40-80% higher, as compared to NAC alone.

3) although, when analyzing some specific differentiation markers, forinstance ALPI, CLDN1 and PCDH1, it was observed that the couplecombinations NAC+SeMet or NAC+Mel had an effect that is comparable tothe combination of the three substances, there is nevertheless a cleartrend to an overall increased induction of differentiation when thethree substances are used in combination.

Example 5: Antibacterial Effect of NAC, SeMet and Mel in HydrogelCoatings of Medical Devices

Methods

Hydrogel Coating:

A method as known in the art for polymer coating of water insolublehydrogel, based on polyurethane (FUR) and polyvinylpyrrolidone (PVP),designed for medical polymeric devices, was used to coat urethral polyvinyl chloride catheters as well as latex catheters. This hydrogel layerhas previously been characterized by the means of the Fourier TransformInfra-Red Attenuated Total Reflection (FTIR-ATR) spectroscopy, staticand kinematic friction factor relative to the uncoated backbone materialand against porcine tissue counter-face, water wetting angle andmicroscopic observations. Tests made by the present inventors (data notshown) confirm changes in surface composition, super-hydrophilicity andlubricity in hydrated state including friction factor reduction. In caseof urethral poly vinyl chloride catheters with hydrogel coated innersurface, the capillary action phenomenon was observed, proving highaffinity between coating and water molecules.

Bacterial Growth and Biofilm Formation:

To measure the effect on formation of adherent sessile bacteria anartificial catheter was constructed according to the technique describedby Nickel and associates (Antimicrob Agents Chemother. 1985;27(4):619-24). Catheter materials used were latex, latex coated withnoble metal comprising hydrogel, latex coated with NAC, SeMet and Mel(NAC+SeMet+Mel) and latex coated with both noble metal and(NAC+SeMet+Mel). The artificial catheter device was connected to a twoliter reservoir, functioning as an in vitro bladder held in 37° C. waterbath. Medium containing Escherichia coli (E. coli) was pumped from thereservoir through the artificial catheter by a pump set to deliver 50ml/h. 10 cm² of the midsection of the catheter was tested before andafter exposure to the E. coli. The strain of E. coli used in theseexperiments was isolated from a patient with catheter associated urinarytract infection. The medium used was artificial urine supplemented with0.4% nutrient broth. The bacteria were stored on a sloping agar in atest tube (slants) at −70° C. and serially cultured at 10 h intervals.Bacterial growth within the in vitro bladder was monitored by usingstandardized turbidity as a growth parameter with a spectrophotometer at600 nm.

Artificial urine containing E. coli was passed through the artificialcatheter for 10 h and the development of bacterial biofilm was monitoredby sampling of catheter material surfaces. Sample discs bearing sessilebacteria were aseptically removed for scanning electron microscopy.Also, quantitative counts of viable adherent bacteria were obtained bylow-output ultrasonication of surface scraping of the catheter disc in asterile phosphate-buffered saline solution. Dilution series were made upto 10⁻⁴ and spread on nutrient agar from which quantitative plate countswere obtained. The catheter specimens designated for SEM were removedfrom the artificial catheter and placed in fixative solution consistingof 5% glutaraldehyde in cacodylate buffer (0.1 M, pH7.2) for 1 h at 22°C., followed by dehydration in a series of aqueous ethanol solutions(20-100% and Freon 113-ethanol solutions (30-100%) and then air dried.Samples were coated with gold in a sputter coater and examined by usinga scanning electron microscope.

Results

The growth on nutrient agar of surface scrapings of the catheter discrevealed that the number of viable E. coli was significantly decreasedon hydrogel coated material compared to uncoated backbone material. Afurther dramatic decrease in the number of adhered bacterial colonieswas noted when adding NAC, SeMet and Mel to the hydrogel.

Number of colonies on the backbone material: 100

Number of colonies on the hydrogel coated material: 73

Number of colonies on the hydrogel with NAC, SeMet and Mel coatedmaterial: 58

Examination by SEM showed that the discs of catheter latex, noble metalcoated latex, (NAC+SeMet+Mel) coated latex, and noblemetal+(NAC+SeMet+Mel) coated latex did not bear significant numbers ofethanol killed bacterial cells before exposure to E. coli. Ten minutesafter exposure to cells of E. coli in artificial urine, significantnumbers of adherent bacterial cells were seen on the latex discs whereasthere was minimal colonization on the noble metal coated latex or(NAC+SeMet+Mel) coated discs and no colonization on the noblemetal+(NAC+SeMet+Mel) coated discs. After 10 h of colonization, thecharacteristic plate-like surface of the uncoated latex discs wascompletely occluded by a large number of adherent bacteria, which wereembedded in large amounts of their own amorphous exopolysaccarides toform a thick adherent biofilm. On the noble metal coated catheter discsand on the (NAC+SeMet+Mel) coated catheter discs there were sparse signsof biofilm formation whereas there was no biofilm on the discs coatedwith the combination of noble metal and (NAC+SeMet+Mel).

The invention claimed is:
 1. A method of administering a hydrogelcoating, the method comprising: exposing the hydrogel coating to bodilyfluids, wherein the hydrogel coating consists of one or more excipientsand active agents consisting of: (i) N-acetyl-L-cysteine; (ii) seleniumin the form of selenomethionine; and (iii) melatonin and/orphysiologically acceptable salts thereof.
 2. A method of treatingdermatological diseases, the method comprising: administering to patientin need thereof, a combination consisting of one or more excipients andactive agents consisting of: (i) N-acetyl-L-cysteine; (ii) selenium inthe form of selenomethionine; and (iii) melatonin and/or physiologicallyacceptable salts thereof.
 3. The method of claim 2, whereinN-acetyl-L-cysteine is administered at a dose of 5-45 mg/kg/day,selenomethionine is administered at a dose of 0.4-1.2 μg/kg/day, andmelatonin is administered at a dose of 0.02-0.08 mg/kg/day.
 4. Themethod of claim 3, wherein N-acetyl-L-cysteine is administered at a doseof 20-30 mg/kg/day, selenomethionine is administered at a dose of 0.8μg/kg/day, and melatonin is administered at a dose of 0.04 mg/kg/day. 5.The method of claim 2, wherein components (i)-(iii) are administereddaily.
 6. The method of claim 2, wherein components (i)-(iii) areadministered simultaneously, sequentially, or by separate combinatorialadministration.
 7. The method of claim 2, wherein components (i)-(iii)are administered orally or epicutaneously.
 8. A method of administeringa hydrogel coating, the method comprising: exposing the hydrogel coatingto bodily fluids, wherein the hydrogel coating consists of one or moreexcipients and active agents consisting of: (i) N-acetyl-L-cysteine;(ii) selenium in the form of selenomethionine; (iii) melatonin; and (iv)dexamethasone and/or physiologically acceptable salts thereof.